Therapeutic proteins and other commercially important polypeptides can be produced from mammalian cells in culture that have been engineered to express high levels of a particular protein or polypeptide of interest. One advantage of producing such proteins in mammalian cell cultures is that they can be designed for secretion, folding and post-translational modifications, such as glycosylation. In such mammalian cultures, control and optimization of cell culture conditions is critical for successful commercial production of proteins and polypeptides. The ultimate amount and quality of protein or polypeptide produced can be significantly affected by the cell culture conditions and reagents.
Supplementation of animal cell culture media with serum generally improves cell viability and also the production of recombinant proteins. Over the past decade or two, regulatory and safety concerns, as well as problems with sourcing heterogeneity, have driven an industry trend toward eliminating serum in commercial production of proteins. Grillberger et al., Biotechnol. J. 2009, 4, 186-201. However, animal cells grown in serum-free media can be very sensitive to nutritional deficiencies which may induce apoptosis. Apoptosis adversely affects both quality and titer of recombinant proteins. Franek and Fussenegger, 2005, Biotechnol. Prog. 21, 96-98. Protein hydrolysates or peptones generated from soy and wheat gluten can sometimes help compensate for the lack of serum. Much effort has been given to identifying the components in these additives, and their optimum concentration ranges, that are responsible for these advantageous effects.
Franek et al. prepared fractions from plant protein hydrolysates and tested them for their ability to support growth and secretion from murine hybridoma cells. Franek et al., 2000, Biotechnol. Prog. 16, 688-692. As an alternative approach, Franek et al. screened available synthetic peptides for their effect on production from a mouse monoclonal cell line in serum-free medium. Franek et al., 2002, Biotechnol. Prog. 18, 155-158. They reported that while single amino acid or dipeptide did not significantly alter the culture parameters, tri-, tetra-, and penta-glycine as well as tri- and tetra-alanine enhanced viable cell density and viability. Id. Certain tri-peptides enhanced production of monoclonal antibody from this mouse monoclonal cell line. Id. In subsequent experiments on CHO cells recombinantly engineered to produce SEAP, this group reported that tetra-glycine increased viable cell density, and that the tri-peptide Gly-Lys-Gly enhanced SEAP production. Franek and Fussengger 2005, Biotechnol. Prog. 21, 96-98.
There remains a need in the art to develop methods for recombinant production of protein in completely defined—media or chemically defined media. While protein hydrolysates can improve survival, they remain a source of heterogeneity. Although certain tri- and tetra-peptides have been reported to improve culture parameters, it is preferable to have as simple and as defined a cell culture process as possible which maximizes recombinant protein titer. Any improvements to recombinant polypeptide expression, titer, cell growth and/or cell viability can lead to higher production levels, thereby reducing costs associated with the manufacture of protein therapeutics. The invention fulfills these needs by providing simple, easy and novel methods of increasing cell growth and protein production.